Bulletin of Insectology 65 (2): 217-222, 2012 ISSN 1721-8861 Plant host differences between Cossus redtenbacheri and Cossus insularis: insights from mechanical tests and molecular phylogeny 1 2 3 1,4,5,6 7 Fredd VERGARA , R. Craig EVERROAD , Guadalupe ANDRACA , Jun KIKUCHI , Hiroshi MAKIHARA 1Advance NMR Metabomics Research Team, RIKEN Plant Science Center, Yokohama, Japan 2RIKEN Advanced Science Institute, Wako, Japan 3Instituto de Ecología, Universidad Nacional Autónoma de México, Mexico 4Biomass Engineering Program, RIKEN, Yokohama, Japan 5Graduate School of Nanobiosciences, Yokohama City University, Japan 6Graduate School Bioagricultural Sciences, Nagoya University, Japan 7Forestry and Forest Products Research Institute, Tsukuba, Japan Abstract Larvae of the family Cossidae are mostly associated with trees. Cossus redtenbacheri (Hammerschmidt) is exceptional in this family because its larvae live in agave. Two approaches were followed to better characterize this host difference. First, a mechani- cal test was performed to evaluate the gallery making ability of C. redtenbacheri and Cossus insularis (Staudinger) larvae trans- ferred to non-natural hosts of differing tissue hardness. Second, the first molecular evolutionary analysis of C. redtenbacheri was performed to assess its relationship with selected wood-feeding Cossidae species. This phylogenetic reconstruction was based on partial sequences of mitochondrial cytochrome c oxidase subunit 1. The agave-feeding phenotype is confirmed as a derived phy- logenetic character within the genus Cossus. The results show that the host difference is not due to mechanical constraints or to phylogentic distinctiveness. The possible types of ecological factors that might explain the host specialization of C. redtenbacheri are discussed. Key words: 28S, borer insects, CO1, Cossus redtenbacheri, Comadia redtenbacheri, Cossus insularis. Introduction on agave instead of trees. A first step in identifying the correct mechanism be- Cossus redtenbacheri (Hammerschmidt) (= Comadia hind the host differences within the Cossidae is there- redtenbacheri) is unusual among the Cossidea (Lepi- fore to test and, if possible, exclude some of the poten- doptera) because its larvae develop in Agavaceae spe- tial mechanisms. Consequently, we have initiated a pro- cies. The larvae of other species in this worldwide fam- ject of investigating agave use by C. redtenbacheri and ily develop in trees and shrubs where they usually bore of feeding niches in the Cossidae. As part of this study galleries in branches and trunks (Davis et al., 2008). we applied two approaches. The first of these was to ex- Anyway, regarding Cossus cossus L. (European goat amine the mechanical boring ability of larvae. If C. red- moth), the larvae can bore artichokes (Inserra, 1962) tenbacheri is able to bore into wood, its specialization and exceptionally sugar bet roots (Ugolini, 1962-63). It on agave is not due to a simple physiological or me- is possible also rear C. cossus on artificial diet (Gavioli chanical inability to bore into wood. Likewise, if Cossus and Baronio, 1987). Agave differs from trees in having insularis (Staudinger), a typical wood boring species, soft rather than hard tissues and different nutrient con- bores into agave, the wood-agave distinction cannot re- tent (Ortiz-Basurto et al., 2008). Agave also has differ- sult from physiological or mechanical factors related to ent chemical defences (Simmons-Boyce and Winston, gallery creation. We therefore used a gallery making 2007). Thus the host-shift of C. redtenbacheri is an in- assay with C. redtenbacheri and C. insularis. teresting ecological question. The second approach was to identify the phylogenetic There is an additional, applied, importance to under- position of C. redtenbacheri within Cossus using a mo- standing the mechanisms underlying the difference in lecular phylogenetic approach. The unique agave-feeding cossid feeding niches. This is because both C. redten- habit of this species would be no surprise if the species is bacheri and agave are culturally and economically im- not, in fact, closely related to other species in the genus. portant in Mexico. The larvae, commonly called gusano We therefore constructed the first molecular phylogeny of rojo de maguey (agave red worm), are used in cooking Cossus that includes C. redtenbacheri using partial se- and the production of spirits (Ramos-Elorduy et al., quences of the mitochondrial cytochrome c oxidase sub- 2011). Larvae for these purposes are currently collected unit 1 (CO1) from 13 Cossidae species. For additional from the wild. This reduces the natural population of the information we also sequenced the nuclear 28S ribosomal species and leads to the destruction of agave and the a- subunit (28S) of C. redtenbacheri and C. insularis. gave habitat. These negative effects would be alleviated Both species were able to bore into wood and agave. by culturing C. redtenbacheri and culturing would be Therefore, mechanical differences do not explain the assisted by knowing how the species came to specialize host specialization. The molecular phylogeny situates C. redtenbacheri well inside both the wood-boring Cos- more cossid species than sequences of any other gene. sidae and the genus Cossus in particular. Thus, the phy- Experimental data have shown that CO1 discriminates logeny does not explain the host specialization. The well between insect species and provides a good phy- agave specialization is thus an ecological feature that logenetic signal (Wilson, 2010). If insect species have requires further explanation. dissimilar CO1 sequences they are very unlikely to be closely related (Smith et al., 2009). In contrast to the mitochondrial CO1, 28S is a nuclear Materials and methods gene. Again, species with dissimilar 28S sequences are unlikely to be closely related. Species where neither Insects CO1 nor 28S are dissimilar are very likely to be from C. redtenbacheri is found mainly in Mexico. Larvae the same genus and certainly within the same clade. were collected from agave plants in the federal state of Hidalgo, central Mexico, in July 2011. C. insularis is DNA extraction, amplification and sequencing distributed entirely within Asia. Its larvae were col- DNA was extracted from 10 larvae of C. redtenbach- lected from willow trees (Salix nipponica Franchet et eri and 10 larvae of C. insularis. None of the larvae Savatier) in Ibaraki prefecture, Japan, in June 2011. used for phylogenetics had been used in gallery making tests but were co-collected from the same natural popu- Gallery making tests lations at the same time and location. The larvae were In order to examine the mechanical ability of the two first stunned by chilling at 4 °C for 10 minutes. Their species, we tested C. redtenbacheri from agave in wood bodies were cleaned by rubbing them against a tissue and C. insularis from wood in agave. C. redtenbacheri soaked with 70% ethanol to remove contaminating was tested in branches of pear trees obtained from a DNA. The larvae were then decapitated and their legs farm close to the site where C. redtenbacheri had been cut off. DNA was then immediately extracted only from collected in Mexico. Pear was chosen because C. insu- the legs using the Qiagen DNEasy® blood & tissue kit laris can live in this tree in Japan (Nakanishi, 2005). (Maryland, USA, cat. 60504) following the manufactur- Tunnels of approximately 10 mm diameter and 100 mm ers protocol for purifying total DNA from animal tissues long were bored longitudinally in branches with a power based on a spin-column. Whole bodies were not ex- drill. These dimensions are within the size range of tun- tracted because Cossidae larvae have been reported to nels bored naturally by C. insularis. Four tunnels were display entomophagy (Ichikawa and Ueda, 2009) and drilled in each of three branches. We removed all loose DNA from other insect species might be present in their wood and sawdust from the tunnels so as to force the guts. The extracted DNA was PCR-amplified using larvae to remove wood themselves. A single larva was 0.5 µL of the forward and reverse primers (50 mM, Op- then introduced into each tunnel, giving a total of 12 eron, table 1), 4 µL dNTPs (2.5 mM each, Takara, Ja- larvae. The tunnel openings were then sealed with caps pan, cat. 4030), 5 µL 10x ExTaq buffer (Takara, Japan, of the same wood. cat. 9152A), 0.25 µL ExTaq (5 U µL-1, Takara, Japan, Agave americana L. plants were purchased from Na- cat. RR01A), 1 µL of the DNA template solution and gashima Shokubutsuen (Kagoshima, Japan). Five leaves 38.5 µL sterile water. PCR thermocycling occurred at were removed from the stem and enclosed together with one cycle of 120 s at 94 °C, five cycles of 40 s at 94 °C, 15 late instar C. insularis larvae in a plastic box. 40 s at 45 °C, and 60 s at 72 °C, followed by 36 cycles The pear branches and the agave leaves were then kept of 40 s at 94 °C, 40 s at 51 °C, and 60 s at 72 °C, with a at room temperature for three weeks during which the final cycle of 300 s at 72 °C (Fisher and Smith, 2008). activity of the larvae was observed. The PCR products were separated by electrophoresis on a 2.5% agarose gel and purified using the QIAquick® Phylogenetic position gel extraction kit (Qiagen, Hilden, Germany, cat. We examined the position of C. redtenbacheri within 28706) following the manufacturer’s protocol for gel the cossid CO1 phylogeny. If its position does not fall extraction using a microcentrifuge. The purified PCR within the clade of wood-boring cossid species then its products were bidirectionally sequenced using the origi- feeding niche is phylogenetically determined. However, nal PCR primers with BigDye v3.1 on an ABI 3730xl if the species lies within the phylogeny then the distinc- DNA Analyzer (Applied Biosystems). tion in feeding niche is not phylogenetic but more likely to be ecological.